1. Field of the Invention
This invention relates to the fields of bacterial molecular biology and recombinant DNA technology.
2. Description of the Related Art
Isomaltulose (6-O-α-D-glucopyranosyl-D-fructofuranose) is a reducing sugar with physical properties and taste very similar to sucrose. It is a structural isomer of sucrose found naturally in honey (Sporns et al., 1992). Isomaltulose has been regarded as an alternative to sucrose with several attractive features: (1) it resists metabolism by cariogenic oral streptococci and does not cause dental decay (Minami et al., 1990); (2) its ingestion leads to very small effects on the concentrations of glucose and insulin in blood, indicating potential as a parenteral nutrient acceptable both to diabetics and non-diabetics (Kawai, et al., 1989); (3) unlike sucrose, it is not fermented by most bacteria and yeasts and is more stable in acid solutions—these properties of isomaltulose facilitate the maintenance of sweetness and taste in fermented foods and beverages (Takazoe, 1989; Schiweck et al., 1991); (4) it does not possess hydroscopic properties like sucrose or lactose—isomaltulose-containing foods are more stable than those containing sucrose (Takazoe, 1989); (5) it selectively promotes bifidobacteria growth among human intestinal microflora (Mizutani, T. 1989). Evidence is accumulating that bifidobacteria are helpful for maintenance of human health and slowing the aging process (Mitsuoka, T. 1990). In Japan isomaltulose has been widely used as a sugar substitute in food (Takazoe, 1989). Biochemical conversion of sucrose for the production of isomaltulose has reached a scale of more than 10,000 tons annually worldwide (Kunz, 1993).
Isomaltulose is also a raw material for the synthesis of surfactants and polymers. With an annual production of around 110 million tons, sucrose is the world's most abundantly produced renewable organic compound. However, because of the acid instability of its intersaccharidic bond and its lacking of specific chemical reaction possibilities, the utility of sucrose as a raw material for the chemical industry has been very limited (Kunz, 1993). Isomaltulose, the reducing isomer of sucrose, on the other hand, can be specifically oxidized and derivatised for production of different chemical products. Isomaltulose has been successfully used for the production of polyamides and polyureas (Kunz, 1993).
Several bacterial species are known to be able to convert sucrose into isomaltulose. U.S. Pat. No. 4,359,531 describes a process for the production of isomaltulose, consisting of immobilizing whole cell or solvent extracts of whole or disrupted cells of Erwinia rhapontici. About 70-95% of sucrose was converted to isomaltulose products. U.S. Pat. No. 4,390,627 describes a method for immobilization of the sucrose mutase enzyme from Protaminobacter rubrum for production of isomaltulose. U.S. Pat. No. 4,670,387 describes a fermentation process to produce isomaltulose using immobilized cells of Erwinia rhapontici, Protaminobacter rubrum, Serratia plymuthica, Esevinia carotovora var atroseptica, Erwinia dissolvens, Serratia merscescens. Again about 70-95% of sucrose was converted. U.S. Pat. No. 4,857,461 discloses a continuous process for the enzymatic preparation of isomaltulose by immobilized crude enzyme from Protaminobacter rubrum, Serratia plymuthica, and Erwinia carotovora. U.S. Pat. No. 5,229,276 and No. 5,336,617 describe a process for preparing trehalulose and isomaltulose with immobilized cells of Pseudomonas mesoacidophila and Agrobacterium radiobacter. 
A single enzyme, isomaltulose synthase (EC 5.4.99.10), is responsible for converting sucrose to isomaltulose. Although several groups of bacteria can convert sucrose to isomaltulose, yields of isomaltulose in the converted products were variable, ranging from 8% to 86% (Tsuyuki et al., 1992; Nagai et al., 1994; Huang et al., 1998). Moreover, these isomaltulose producing strains not only transform sucrose to isomaltulose and trehalulose but also produce about 2-7% of glucose as by-products (McAllister et al., 1990; Tsuyuki et al., 1992; Huang et al., 1998). This is a considerable industrial problem because elaborate purification procedures are necessary to remove these contaminating compounds (Sugitani et al., 1993, U.S. Pat. No. 5,229,276). Some strains are even capable of hydrolysis of isomaltulose into glucose and fructose.
More recently, U.S. Pat. No. 5,786,140 disclosed isolation of genes of sucrose isomerase from several organisms. By using a DNA probe based on the amino acid sequence deduced from the N-terminus of purified sucrose isomerase, the genes for sucrose isomerase were cloned from Protaminobacter rubrum, and Enterobacter sp. Partial DNA sequences for sucrose isomerase were further isolated from E. rhapontici and P. mesoacidphila by utilizing PCR.